The enzyme .beta.-galactosidase has for a long time found wide use in enzyme-linked immunosorbent assays (ELISA) (Engvall and Perimann, 1971, Immunochemistry 8:871) and homogeneous substrate labeled assays (Burd et al., 1977, Clin. Chem. 23:1402). In addition, .beta.-galactosidase forms the basis of a widespread genetic system for DNA cloning and DNA sequencing (Messing, 1983, Methods of Enzymology 101:20).
.beta.-Galactosidase is a tetrameric protein having a molecular weight (MW) of about 540,000 daltons. The four identical monomers consist of 1023 amino acids, each with a MW of 116,000 daltons. The monomeric protein, as shown in FIG. 1, is divided into three regions: (1) the N-terminus proximal segment (the .alpha.-region), (2) a middle region, and (3) a C-terminus distal segment (the .omega.-region).
Mutant polypeptides derived from .beta.-galactosidase are known which can complement or spontaneously restore enzyme activity when added to extracts of appropriate .beta.-galactosidase negative mutants. This phenomenon is known as intracistronic complementation. When complementation involves a deletion or chain interruption in the .alpha.-region, the process is referred to as .alpha.-complementation. An example of .alpha.-complementation is provided by the M15/CNBr2 complementation system. The M15 mutant polypeptide lacks amino acids 11-41 of .beta.-galactosidase and exists in solution as an enzymatically inactive dimer. A polypeptide derived from .beta.-galactosidase by cyanogen bromide cleavage, denoted the CNBr2 peptide (CNBr2), consists of amino acids 3-92. CNBr2, when mixed with the dimer M15, promotes spontaneous reconstruction of the .beta.-galactosidase tetramer with full enzymatic activity (Langley and Zabin, 1976, Biochemistry 15:4866). The M15 peptide is therefore known as an .beta.-acceptor and CNBr2 as an .beta.-donor.
While this represents a well-studied complementing system, CNBr2 can also serve as an .alpha.-donor for the M112 dimer, a deletion of amino acids 23-31 within .beta.-galactosidase (Lin, Villarejo and Zabin, 1970, Biochem. Biophys. Res. Common. 40:249; Celeda and Zabin, 1979, Biochem. 18:404; Welply, Fowler and Zabin, 1981, J. Biol. Chem. 256:6804; Langley et al., 1975, Proc. Natl. Acad. Sci. USA 72:1254). In the example of complementation of M15 by CNBr2, amino acid sequences 3-10 and 42-96 are both present in duplicate in the enzymatically inactive complex.
Intracistronic complementation also occurs at the C-terminus of .beta.-galactosidase (the .omega.-region). An early publication that describes structural studies related to complementation of .beta.-galactosidase in the carboxy-terminal region is M. E. Goldberg, The Lactose Operon, Cold Spring Harbor Laboratory, 1970. The most widely studied example is the X90-acceptor peptide that deletes that last 10 amino acids, 1013-1023. The X90 peptide exists as a monomer and can be complemented by CNBr24, a cyanogen bromide digestion product of .beta.-galactosidase consisting of amino acids 990-1023, to reform enzymatically active tetramer (Welphy et al., 1980, Biochem. Biophys. Res. Common. 93:223).
A number of patent applications and patents relating to complementation assays have arisen out of the laboratories of the present inventors. Those applications that are directed to .beta.-galactosidase enzyme donors and acceptors are U.S. application Ser. No. 666,080, filed Oct. 29, 1984, now abandoned; U.S. application Ser. No. 721,267, filed Apr. 8, 1985, now U.S. Pat. No. 4,708,929; U.S. application Ser. No. 788,370, filed Oct. 22, 1985, now U.S. Pat. No. 5,120,653; U.S. application Ser. No. 347,679, filed May 5, 1989, now abandoned; U.S. application Ser. No. 410,996, filed Sep. 22, 1989, now abandoned; and PCT application PCT/US90/02491, filed May 4, 1990, now pending as an international PCT application designating the US. All of these patents and patent applications are herein incorporated by reference. In particular, several of these earlier patents and applications disclose the complete genetic and amino acid sequence of the beta-galactosidase enzyme.